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HEMATOPOIESIS
From the Department of Cell Biology, University of
Groningen, the Netherlands; Division of Hematology/Oncology, Blood and
Marrow Transplant Program, Lucille P. Markey Cancer Center, and
Department of Physiology, University of Kentucky Medical Center,
Lexington, KY.
We have previously demonstrated that young adult DBA/2 (DBA) mice
have more stem cells than C57BL/6 (B6) mice, as measured in a
cobblestone area-forming cell (CAFC) assay using unfractionated marrow.
To study the nature of this difference, we have now compared the
proliferative fate of single, highly enriched
Sca-1+c-kit+Lin Hematopoietic stem cells have the potential to
produce mature blood cells of at least 8 different lineages. This
impressive proliferative capacity is most convincingly demonstrated by
the ability of a single intravenously transplanted stem cell to fully reconstitute the hematopoietic system of a lethally irradiated mouse.1-5 Although several studies suggest that stem cell
potential is subject to replicative erosion during ontogeny and
aging,6-9 in the mouse, stem cells can be expanded in vivo
to an extent that is unlikely to be encountered during normal
physiology.10-12 These developmental properties have paved
the way for the application of hematopoietic stem cells in a wide
variety of clinical situations. Most notably, stem cells harvested from
the bone marrow or neonatal and adult peripheral blood can be
cryopreserved and used for hematopoietic rescue in allogeneic and
autologous transplantation protocols. Stem cell transplantation after
high-dose chemotherapy has enabled a significant improvement in the
cure of patients affected by a variety of malignancies.13
However, the rate of recovery of circulating blood cells in
transplanted patients is highly variable, and even with substantial
growth factor support some patients fail to normalize blood cell counts
in a clinically acceptable time frame.14,15 Undoubtedly,
this heterogeneity results partly from prior cytotoxic treatment,
disease status, and the number of cells infused,14 but
even in patients that have undergone treatments of apparent equal
intensity, wide variations in hematopoietic response are not unusual.
These data suggest that patient-to-patient variation in hematopoietic
recovery after stem cell transplantation may have a genetic basis.
Indeed, the heterogeneity in patient responses to therapeutic agents in
general, and the genetic basis of this variation, has established
the nascent field of pharmacogenomics, which is propelled by
advances made in the Human Genome Project.16
The involvement of genetic factors in the regulation of hematopoietic
cell production is perhaps most compellingly demonstrated in a series
of preclinical mouse studies, partly performed in our own
laboratories.17-23 We have shown that the frequency of hematopoietic progenitors and stem cells in unfractionated marrow from
DBA/2 (DBA) and AKR/J mice is higher than in all other strains tested,
including C57BL/6 (B6).20 We have demonstrated that DBA
progenitor cells have a higher proliferative activity than B6
cells,17,21 and embryo-aggregation studies have suggested that this is a cell-intrinsic trait.18
In this study, we have investigated in more detail the functional
activity of DBA and B6 stem cells. We compared the in vitro growth
kinetics of single, highly enriched
Sca-1+c-kit+Lin Mice
Enrichment of hematopoietic stem cells
Measurement of in vivo hematopoietic reconstitution kinetics The 1000 enriched B6 or DBA stem cells were intravenously injected into 21 (Ly-5 congenic) B6.SJL or 33 syngeneic DBA hosts, respectively. Recipient mice were exposed to 9 Gy total body -irradiation administered in 2 doses of 4.5 Gy approximately 3 hours
apart just before transplantation. Mice were bled from the
retro-orbital sinus 6, 9, 12, 15, 18, 25, 32, and 42 days later. Until
day 25, only half of the mice in each cohort was analyzed alternately at each time so that no individual animal was bled more frequently than
every 7 days. Circulating leukocyte, erythrocyte, and platelet counts
were measured by analysis of 40 µL of blood using a System 9118+
Hematology Series Cell Counter (BioChem ImmunoSystems Inc, Allentown, PA).
Administration of 5-fluorouracil Mice were subcutaneously injected with 5-FU (Fluracedyl, Pharmachemie, Haarlem, The Netherlands) at a dose of 200 mg/kg in saline. Three mice were killed per timepoint. Mice were bled from the retro-orbital venous plexus, and white blood cells were quantified using an automated cell analyzer (Coulter Model ZF, Coulter Electronics Ltd, Dunstable, Beds, England). Hematocrit values were calculated after centrifugation of approximately 100 µL of anticoagulated blood in microcapillary tubes.Hematopoietic cell assays The CAFC assay was performed exactly as detailed previously.20 In short, confluent monolayers of FBMD-1 stromal cells were established in 96-well tissue culture-treated plates. After 10 to 14 days, wells were seeded either with sorted Sca-1+c-kit+Lin cells at a dose
of 1, 3, 10, or 30 cells per well using an automated cell deposition
unit, or with unfractionated marrow at a dose of 81 000,
27 000, 9 000, 3 000, 1 000, or 333 cells per well. Typically 30 replicate wells per dilution were evaluated. However, in experiments
with sorted cells 120 replicate wells were seeded with 1 cell, 60 wells
with 3 or 10 cells, and 10 to 30 replicate wells were seeded with 30 cells. Individual wells were screened every 3 to 4 days for the
presence of a "cobblestone area," defined as a colony of at least 5 cells growing underneath the stroma, according to
definitions established by Ploemacher et al.24 Frequency
estimates were calculated using maximum likelihood
analysis,25 considering at least 3 cell doses that yielded
both negative and positive wells.
Cobblestone area-forming cell frequencies in purified DBA and B6
Sca-1+c-kit+Lin cells isolated
from the bone marrow of both strains. Two independent sorting
experiments were performed. In the first experiment, bone marrow was
harvested from a single 10-month-old mouse of each strain. In the
second sorting experiment, marrow cells were pooled from 3 8-week-old
mice of each strain. The immunophenotype of both young and old,
as well as of DBA and B6 cells, was very comparable, and thus the same
sorting gates were used throughout the study. Six percent to 12% of
marrow cells were lineage negative. Approximately 0.3% to 0.5% of the
Lin cells expressed both c-kit and Sca-1, and this
fraction was sorted and evaluated for CAFC activity. Sorted cells were
seeded in a limiting-dilution fashion (1, 3, 10, or 30 cells per well)
onto preestablished stromal cell layers in 96-well plates, as described in the "Materials and methods." Figure
1 depicts the CAFC frequency as a
function of culturing time. DBA cells are compared with B6 cells
obtained from young (Figure 1A) or old (Figure 1B) donors. It is
evident that the general pattern for both experiments was very similar.
During the first 9 days of culture, no cobblestone areas were detected
in any well, indicating that the stringent selection criteria that were
used in the sorting protocol depleted the most mature progenitor cells
that typically appear between days 5 and 7.20,24 In young
animals, CAFC activity in the
Sca-1+c-kit+Lin fraction peaked
at day 14 in B6 mice, and at day 21 in DBA mice, reaching values in
both strains of approximately 104 CAFC per 105
(ie, 10%) sorted cells (Figure 1A). CAFC frequency in B6 sorted cells
decreased rapidly thereafter and no positive wells were detected after
day 35. In contrast, assay of sorted DBA cells showed that the decline
in CAFC frequency was slower, and some wells remained positive even
after day 50. We have previously measured CAFC activity in
unfractionated marrow from these strains, and found that the frequency
of CAFC day 35 is approximately 1 per 105 B6 cells and
approximately 3 per 105 DBA cells.26 The
current experiments reveal that this variation in stem cell frequency
is more pronounced when purified cells are studied. Table
1 compares CAFC frequencies in
unfractionated marrow and the stem cell-enriched fraction from young
mice. CAFC day 35 frequency was 497 per 105 cells for
sorted B6 cells, and 6172 per 105 cells for sorted DBA
cells, ie, the frequency of this stem cell subset was more than 10-fold
higher among Sca-1+c-kit+Lin DBA
cells than in phenotypically identical B6 cells.
Intrastrain comparisons of young and old mice, as shown in Figure 1C and D for B6 and DBA respectively, revealed that late-appearing CAFC activity in sorted cells from old mice was higher than in cells from young mice. This is in agreement with our previous finding when we quantified CAFC subsets using unfractionated marrow from young and 1-year-old mice.20,27 In addition, CAFC activity of old cells could be detected at later timepoints (more than 50 days) than that of young cells. The fate of single, purified DBA and B6
Sca-1+c-kit+Lin cells from both
young and old DBA and B6 mice in the CAFC assay system. Every 3 to 4 days, all 480 (4 × 120) wells were screened for cobblestone activity
to track the proliferative fate of each individual cell. Figures
2 and 3
show the results for young B6 and DBA cells, respectively. The data
obtained for single sorted cells from 10-month-old mice were very
similar (data not shown).
Of a total of 120 young B6 stem cells, 29 (ie, 24%) gave rise to a cobblestone area at some time during the culture period. For old cells, this number was 43 per 120, ie, 35%. The first wells4 became positive at day 9, and the last positive well was observed at day 34. Colonies persisted for a maximum interval of 3 observation points (ie, approximately 8 days). In contrast, when the 120 DBA stem cells were evaluated, major differences compared with B6 cells were observed (Figure 3). The total number of cells with cobblestone area-forming potential was 64% or 53%. For old DBA cells, this number was 66% or 55%. No well with visible hematopoietic activity was detected before day 13. Although only a minority (3 of 29) of the B6 colonies remained for a maximum of 3 timepoints, most DBA colonies persisted for at least this long, and clone C10-1 remained for 8 timepoints (approximately 25 days). Overall, Sca-1+c-kit+Lin Engraftment kinetics after transplantation of 1000 purified DBA and
B6
Sca-1+c-kit+Lin cells into
syngeneic and congenic recipients, respectively, and monitored the
regeneration of circulating leukocytes, erythrocytes, and platelets for
2 months (Figure 4). White and red blood
cell counts in mice transplanted with enriched DBA stem cells recovered to normal values in approximately 12 days (Figure 4A and B). Leukocyte engraftment was biphasic, so counts did drop transiently to
approximately 50% of normal on day 15 before recovering permanently by
day 32. In contrast, mice transplanted with enriched B6 stem cells
showed delayed recovery as blood cell counts failed to normalize until approximately 30 to 40 days. Platelet counts recovered significantly faster in DBA-stem cell-transplanted recipients as well, but the effect was less pronounced as for the other blood cell lineages (Figure 4C).
Stem cell pool size correlates with hematopoietic recovery rate after myeloablation Finally, we compared the tempo of recovery of several hematopoietic parameters in DBA and B6 mice after administration of a single dose of 5-FU (200 mg/kg). Figure 5A depicts the number of various CAFC subsets surviving 24 hours after 5-FU administration in DBA and B6 mice. Because of the larger total stem cell pool in normal DBA mice (Table 1), the absolute number of all CAFC subsets that survived 5-FU treatment was higher in this strain. Next, the recovery kinetics of the bone marrow CAFC day-7 and day-35 compartments was determined from days 1 to 14 after 5-FU. CAFC day-7 numbers recovered approximately twice as fast in DBA mice than in B6 mice (Figure 5B). At day 7, the number of CAFC day-7 numbers in B6 mice was still less than 5% of normal, whereas progenitors in DBA mice had already normalized. Similarly, the recovery kinetics of more primitive CAFC day 35 were very different in the 2 strains (Figure 5C). In B6 mice, stem cell numbers showed a nadir between 2 and 7 days after 5-FU treatment, and exhibited a profound overshoot of normal numbers around day 10. Stem cells in DBA mice were also reduced initially, but to a much lesser extent than in B6, and, perhaps as a result, the overshoot was less dramatic.
To determine whether these CAFC recovery differences were of any
relevance for mature blood cell production, we also measured peripheral
blood cell counts in these same mice. Figure
6A and B show hematocrit values and white
blood cell counts, respectively. During the first week after 5-FU, no
significant differences were detected between the 2 strains. However,
consistent with the pattern of progenitor cell recovery shown in Figure
5B, hematocrit values began to recover faster in DBA mice than in B6
mice, and DBA leukocyte counts exhibited a strong overshoot that was
not observed in B6 animals.
Variation in the frequency, proliferative activity, and mobilization potential of mouse and human hematopoietic stem cells is well described.20-23,26,28 Clinically, this is manifested in a multitude of parameters, ranging from differences in response to hematopoietic growth factors, to hemotoxicity after chemotherapy and speed of recovery after stem cell transplantation. Elucidation of the mechanisms contributing to such variation are of significant importance to the field of stem cell biology and transplantation. To address these issues, we have previously focused on 2 strains of mice that possess widely disparate hematopoietic stem cell characteristics; B6 mice have relatively few stem cells with a low proliferative rate, whereas DBA mice have several-fold more cells with a significantly higher cycling activity.20-22,26 Chromosomal loci have been identified that are involved in some of the traits in which these 2 strains differ.21,22,26 In parallel with genetic approaches ultimately directed at identifying genes involved in this variation, this study was conducted to characterize in detail differences in the functional behavior in vitro and in vivo of stem cells from these 2 model strains. DBA mice have been shown to have a much larger late-appearing CAFC and
LTC-IC compartment than B6 mice.22,26 Therefore, we anticipated that the number of
Sca-1+c-kit+Lin Our data reveal not only quantitative differences between these 2 inbred mouse strains (ie, larger stem cell pool size in DBA mice), but
also distinct qualitative effects (ie, single DBA stem cells exhibit
more "primitive" growth kinetics in vitro than B6 stem cells). In
keeping with these in vitro observations, lethally irradiated recipient
mice transplanted with 1000 DBA
Sca-1+c-kit+Lin In summary, it may be of significant clinical value to identify, prospectively, patients with a relatively small stem cell compartment who may experience more severe hemotoxicity during cancer therapy. Our study underscores this point in a preclinical mouse model, and offers a first approach to address this issue experimentally.
Submitted November 5, 1999; accepted April 13, 2000.
Supported by funds provided by NIH grants RO1 AG16653 to G.V.Z. and RO1 HL61392 to S.J.S., the Lucille P. Markey Cancer Center, the University of Kentucky Hospital and the Department of Internal Medicine. G.d.H. is a fellow of the Netherlands Organization for Scientific Research (NWO) and of the Royal Netherlands Academy of Arts and Sciences (KNAW). S.J.S. is the recipient of a Junior Faculty Scholar Award from the American Society of Hematology.
G.d.H. and S.J.S. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Gerald de Haan, Department of Cell Biology, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands; e-mail: g.de.haan{at}med.rug.nl.
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 F. Martelli, B. Ghinassi, B. Panetta, E. Alfani, V. Gatta, A. Pancrazzi, C. Bogani, A. M. Vannucchi, F. Paoletti, G. Migliaccio, et al. Variegation of the phenotype induced by the Gata1low mutation in mice of different genetic backgrounds Blood, December 15, 2005; 106(13): 4102 - 4113. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) hemopoietic stem cells.
J Immunol.
1996;156:3207-3214